Protocol to reconstitute translationally arrested heat shock mRNPs and condensates in vitro

Summary Heat shock (HS) coincides with the assembly of translationally arrested heat shock messenger ribonucleoprotein particles (HS-mRNPs) and condensates. Here, we present a protocol to reconstitute HS-mRNPs and HS condensates with eIF4G, eIF4E, Pab1p, and mRNA in vitro. In addition, we describe the necessary steps to measure the effect of HS-mRNPs and HS condensates on translation in yeast extracts. The protocol can be modified to study mRNPs and condensates assembled with other proteins and to study translation in extracts prepared from different cells. For complete details on the use and execution of this protocol, please refer to Desroches Altamirano et al.1


Potassium acetate 100 mM
Magnesium acetate 3 mM

DTT 1 mM
Prior to the addition of Mannitol, adjust the pH of the buffer to pH 7.5 with KOH.The buffer is stable for 6 months at 4 C. DTT is added directly before use and once added, the buffer is stable for 1 day at 4 C.The pH of the buffer with all components is adjusted to pH 7.5 with HCl.DTT is added directly before use and once added, the buffer is stable for 1 day at $23 C. STAR Protocols 5, 103275, September 20, 2024

Protocol
Note: Avoid thawing and freezing of the reaction mix.The activity of CPK in the reaction mix decreases when stored for longer than 3 months at À80 C.

STEP-BY-STEP METHOD DETAILS Purification of eIF4G and Pab1p
Timing: 2 weeks eIF4G and Pab1p are recombinantly expressed and purified from baculovirus-infected Sf9 cells (Figures 1A and 1B).eIF4G and Pab1p are expressed as fusion constructs with maltose binding protein (MBP) placed at the N-terminus.Fusion MBP-eIF4G and MBP-Pab1p are captured using amylose resin and further purified using size exclusion chromatography (SEC) (Figures 1C  and 1D).f.Next day, add 1 mL of 4% PBS with 2% Pen-Strep (this will result in $2% FPS with $1% Pen-Strep).g.Place sealed plate at 27 C with shaking at 200 rpm for 3 days.h.Collect cells at 500 3 g for 5 min at $23 C. i. Collect 1.5 mL of supernatant.This contains the released P1 virus.j.To generate P2 virus, add 50 mL of P1 virus to 50 mL of 0.5 3 106 Sf9 cells.k.Add FBS and Penicillin-Streptomycin to a final concentration of 2% and 1% respectively.l.Place at 27 C with shaking at 200 rpm for 5 days.m.Collect cells at 2,000 3 g for 5 min at $23 C. n.Collect the supernatant.This contains the released P2 virus.o.Store P2 virus at 4 C and protected from light.
i. eIF4G retention volume: $65 mL ii.Pab1p retention volume: $75 mL CRITICAL: Do not pool fractions with eIF4G or Pab1p that elute close to the void volume of the column.While these fractions contain eIF4G and Pab1, they also contain a high contamination of nucleic acids and irreversibly aggregated proteins (Figures 1E and 1F).Pooling fractions with aggregated and/or nucleic acid contamination will affect downstream experiments, such as the assembly of mRNPs and condensates.
7. Store purified eIF4G and Pab1p.a. Measure protein concentrations in pooled fractions with A 280 and concentrate proteins with Amicon Ultra Centrifugal Filters with 30 kDa molecular weight cutoff to a final concentration of $80 mM for eIF4G and $200 mM for Pab1p.Centrifuge at 4,000 3 g at 20 C. i. Extinction coefficients of eIF4G and Pab1p are 73,020 M À1 cm À1 and 39,895 M À1 cm À1 respectively.
Note: The final yield of purified eIF4G and Pab1p from 1 L of cells is $2 mg and $4 mg, respectively and have a 260/280 nm ratio of $0.5-0.6,indicating little nucleic acid contamination.
b. Store proteins in 5-10 mL aliquots in PCR tubes.c.Flash-freeze in liquid nitrogen and store at À80 C.

Purification of eIF4E
Timing: 3 days eIF4E is recombinantly expressed and purified from chemically competent BL21-AI E. coli cells (Figures 2A and 2B).eIF4E is expressed as a fusion construct with maltose binding protein (MBP) placed at the N-terminus.In this case, MBP is not utilized for affinity capture on amylose resin but to increase the solubility of eIF4E and improve the yield of purified protein.MBP-eIF4E is captured on resin covalently modified with m 7 GTP prior to SEC (Figure 2C).
To prevent the purification of other cap-binding proteins, we recommend purifying eIF4E from E. coli.
8. Express eIF4E in BL21-AI cells.a. Transform BL21-AI chemically competent cells with plasmid encoding MBP-eIF4E.i. Select cells by plating on LB agar plate with 50 mg/mL kanamycin.
CRITICAL: Make sure that the yeast coding sequence of eIF4E is codon optimized for expression E. coli.Utilize online tools, such as the Codon Optimization Tool of IDT and chose Escherichia coli K-12 for codon optimization of your sequence.If not codon optimized, the protein is prone to degradation when expressed in BL21-AI E. coli due to codon bias and the synthesis of incomplete or misfolded proteins.
b. Inoculate a single colony into 20 mL of LB with 50 mg/mL kanamycin and place at 37 C with shaking at 180 rpm for $16 h.c.Next day, dilute the culture in 500 mL of LB with 50 mg/mL kanamycin to an optical density or absorbance at 600 nm (A 600 ) of 0.05.d.Place at 37 C and grow to an optical density A 600 of 0.5-0.6 with shaking at 180 rpm.e. Cool down the culture by placing it at 20 C with shaking at 180 rpm for 30 min.f.Induce protein expression with 0.1 mM IPTG and 0.2% L-arabinose.

Note:
The expression of T7 RNA polymerase in Bl21 AI cells is under control of araBAD promoter and induced with the addition of L-arabinose.This allows for tight control of gene expression and represses leaky expression of the protein of interest.g.Place the culture at 20 C with shaking at 180 rpm for $16 h. 9. Harvest and lyse cells.
a. Harvest cells by centrifugation at 4,000 3 g for 10 min at $23 C. b.Add one cOmplete protease inhibitor cocktail tablet and 0.1 U/mL benzonase to 100 mL Buffer E and place on ice.c.Resuspend cell pellet with 100 mL of cold Buffer E supplemented with a cOmplete protease inhibitor cocktail tablet and 0.1 U/mL benzonase (step 9b).
Note: Use buffer with low salt concentrations at all purification steps, because eIF4E can irreversibly aggregate at high salt concentrations (>500 mM KCl).
d. Lyse cells with a LM10 pneumatic microfluidizer processor cooled with ice.i. Lyse cells with a pressure of 12,000 pound-force per square inch (psi).
ii. Repeat the previous step for complete lysis of cells.e. Clear the lysate with centrifugation at 75,600 3 g for 30 min at 4 C using rotor JA 25.50 and Beckman Coulter Avanti J-25 High Speed Centrifuge.10.Affinity capture of MBP-eIF4E.
a. Transfer the supernatant of the cleared lysate to 3 mL of Immobilized g-Aminophenyl-7-Methyl-guanosine-5 0 -triphosphate (m 7 GTP) (C10-spacer) resin equilibrated in Buffer E. b.Incubate for 1 h at 4 C with 10 rotations/min.CRITICAL: We strongly recommend using m 7 GTP resin for the capture of eIF4E.When not bound to m 7 GTP, eIF4E is prone to irreversibly aggregate at high concentrations (see troubleshooting problem 2).Affinity capture with amylose or nickel resin will result in the irreversible aggregation of eIF4E due to the high concentration of captured eIF4E on the resin.Alternatively, eIF4E can be captured with amylose or nickel resin, but m 7 GTP sodium salt should be added to the buffer to stabilize eIF4E.Note: m 7 GTP sodium salt absorbs at 280 nm.Measure protein concentrations using colorimetric methods, such as with the Bio-Rad Protein Assay.
Note: Typical yield of eIF4E at this step is $3.5 mg from 500 mL of cells.
a. Concentrate elution to $400 mL using Amicon Ultra Centrifugal Filters with 3 kDa molecular weight cutoff.Centrifuge at 4,000 3 g for $20 min at 20 C. b.Filter eIF4E elution with 0.22 mm PES syringe filter to remove aggregates.c.Load eIF4E elution onto a Superdex 200 Increase 10/300 GL column equilibrated in Buffer E using an A ¨KTA Pure Protein Purification System with a multi-wavelength UV-Vis monitor (measure A 280 and A 254 ).d. Fractionate the elution in 0.2 mL fractions.e. Check fractions with SDS-PAGE using 15% acrylamide gels (Figure 2C).f.Pool fractions containing eIF4E (Figure 2D).
i. eIF4E elution volume: $15 mL Note: If too concentrated, a fraction of eIF4E may be aggregated but can be separated by SEC.The aggregated fraction appears as a left-shoulder in the chromatogram (Figure 2D).If this is the case, take fractions that exclude this shoulder.
13. Store purified eIF4E.a. eIF4E can be flash-frozen in liquid nitrogen and stored at À80 C directly after SEC.i.Typically, the concentration of eIF4E in elution fractions from SEC is $50 mM.b.Alternatively, eIF4E can be concentrated to 100 mM with 100 mM m 7 GTP sodium salt with Amicon Ultra Centrifugal Filters with 3 kDa molecular weight cutoff prior to freezing and storage.Centrifuge at 4,000 3 g at 20 C for 5 min.Monitor protein concentration using colorimetric methods, such as with the Bio-Rad Protein Assay.

Note:
The final yield of purified eIF4E from 500 mL of cells is $3 mg.
c. Store proteins in 5-10 mL aliquots in PCR tubes.d.Flash-freeze in liquid nitrogen and store at À80 C.

In vitro transcription of mRNA
Timing: 2 days b.Check PCR product with a 1% agarose gel.The size of The PCR product is 830 bp (Figure 3B).c.Purify the PCR product using a Monarch PCR & DNA purification kit.
Note: Other DNA and PCR clean-up kits can be used.
Note: Please refer to the manufacturer's protocol (mMESSAGE mMACHINE Kit).Alternative kits can be used.However, make sure that the kit contains mRNA capping enzymes or that capping enzymes are supplemented.
a. Combine reagents in the following order at $23 C: Note: Aminoallyl-UTP-ATTO-488 can be added to synthesize fluorescently labeled mRNA.Cleaned PCR template (0.3-0.5 mg total) 6
Note: A 30 min incubation results in the addition of 100-200 A nucleotides to mRNA (Figure 3C).For longer poly(A) tails, increase the incubation time.i.Typical concentrations of mRNA are $1,000-1,500 ng/mL or 2-3 mM.k.Store mRNA in 5-10 mL aliquots in PCR tubes.l.Flash-freeze in liquid nitrogen and store at À80 C.
Note: mRNA can be thawed and re-frozen.However, limit freeze and thawing to three times because the mRNA may start to aggregate (see troubleshooting step 5).Cell-free extracts for in vitro translation assays are prepared from exponentially growing S. cerevisiae cells (Figure 4).Here, we outline a protocol that was adapted from Wu and Sachs 2014, 3 starting with a 1 L culture of yeast cells and yielding $5 mL of yeast extract.
19. Grow S. cerevisiae cells to exponential phase.a. Inoculate S. cerevisiae strain W303 in 20 mL of YPD and place at 30 C with shaking at 200 rpm for $16 h.
Note: Other strains of S. cerevisiae can be used to prepare extracts.In addition, genetically modified strains (with gene deletions or expressing fluorescently labelled proteins) can be used.
b. Next day, dilute cells to an optical density A 600 of 0.2 in 1 L of YPD and place at 30 C with shaking at 200 rpm for $6 h or until the culture reaches an optical density A 600 of 1.0-1.2.20.Harvest and lyse cells.
Note: All following steps should be done on ice or at 4 C. We recommend that all following steps are done in succession.f.On the last spin, centrifuge cells at 3,000 3 g for 4 min at 4 C. g.Remove the supernatant and weigh the cell pellet.
i. Typical weight of cell pellet from 1 L of cells is $3 g.h.Add one tablet of EDTA-free cOmplete protease inhibitor to 40 mL of Buffer Y/M and place on ice.i. Resuspend the cell pellet with 1.5 mL of cold Buffer Y/M supplemented EDTA-free cOmplete protease inhibitors per gram of cell weight.i.For a cell pellet of 3 g, resuspend the pellet in cold 4.5 mL of Buffer Y/M supplemented EDTA-free cOmplete protease inhibitors.
CRITICAL: Make sure to use EDTA-free protease inhibitors because EDTA dissociates ribosomes and can inhibit the activity of enzymes in the extract.j.Combine 800 mL of resuspended cells with cold 500 mL of acid-washed glass beads (425-600 mm) in pre-chilled 1.5 mL tubes.Repeat this step for the remainder of resuspended cells.k.Lyse cells by beat beating using a Mixer Mill MM 400 set to 25 oscillations per second for 20 min at 4 C. l.Centrifuge tubes at 650 3 g for 2 min to remove whole cells and reduce foam.m.Transfer the supernatant to pre-chilled centrifuge tubes and centrifuge at 21,000 3 g for 10 min at 4 C to remove cellular debris.n.Transfer the supernatant to a Zeba spin desalting column with a 7 kDa molecular weight cutoff equilibrated with cold Buffer Y. o.Centrifuge at 215 3 g for 3 min at 4 C. 21.Nuclease treatment and storage.
Note: Below, we describe an example procedure for the reconstitution of non-heated and HS-mRNPs.Different combinations of proteins, mRNA, buffers, and heating temperatures can be used for comparative analyses.a.In a 1.5 mL tube, combine eIF4G, eIF4E and Pab1p to a 23 concentration.
Note: Use low-binding tubes to reduce the adsorption of proteins and mRNA to the walls of tubes.b.To a 1.5 mL tube, dilute mRNA to a 23 concentration.c.In a 1.5 mL tube, combine protein and mRNA solutions in a 1:1 volume ratio.d.For the reconstitution of HS-mRNPs, place the protein-mRNA sample in a ThermoMixer for 10 min at 45 C.

Note:
We recommend that additional analyses are utilized to characterize the reconstituted mRNPs.For example, we recommend measuring the size distribution of particles using dynamic light scattering (DLS).
e. Confirm the reconstitution of mRNPs with DLS using the Prometheus Panta.i. Load 10 mL of sample into high-sensitivity capillaries.
ii. Perform a high sensitivity size analysis at 25 C.
Note: Below, we describe an example procedure for the reconstitution of condensates heated at different temperatures.In this case, we prepare protein-mRNA samples at a five-fold higher concentration, so that the final concentration of proteins in in vitro translation (IVT) assays in step 28 is 750 nM.Different combinations of proteins, mRNA, buffers and heating temperatures can be used for comparative analyses.a.In a 1.5 mL tube, combine eIF4G, eIF4E, Pab1p and mRNA at a 53 concentration.

EXPECTED OUTCOMES
Examples of data from IVT assays is shown in Figures 6B-6D.Typically, relative luminescence unit (RLU) measurements in IVT assays with PAB1-NLuc mRNA are in the range of 2,000-5,000 counts/s (Figure 6B).When the mRNA is associated with HS-mRNPs or HS-condensates, the RLU decreases (Figures 6C and  6D), demonstrating that the mRNA is translationally repressed and inaccessible to the ribosomes in the extract.The raw data can be normalized to demonstrate the fold-change in translation (Figure 6D).

LIMITATIONS
The assembly of translationally arrested HS-mRNPs and HS-condensates necessitates a strong association between the mRNA and translation factors.Additional experiments, including the monitoring of the apparent binding and dissociation of proteins to mRNA are thus necessary.As an example, refer to Desroches Altamirano et al., 2024. 1 Cellular constituents may be depleted during the preparation of yeast extracts.We have noticed that in the absence of EDTA in the lysis buffer, some translation factors and ribosomal constituents are depleted from the supernatant after step 20m.Irrespective of this limitation, RLU measurements between extract preparations are reproducible (Figure 6B).
We would also like to emphasize that yeast extracts already contain the proteins supplemented in mRNPs and condensates (e.g., eIF4G, eIF4E and Pab1p).However, this protocol is utilized as a comparative study of the accessibility of mRNA associated in mRNPs and condensates reconstituted in vitro, as demonstrated in Desroches Altamirano et al., 2024. 1 Alternatively, extracts prepared from cells with gene knockouts or depleted for proteins of interest can be utilized.

TROUBLESHOOTING Problem 1
Yield of purified eIF4G is low.

Protocol Potential solution
Increase the volume of insect cells to 2 L. Monitor the expression of MBP-eIF4G in insect cells over 3 days by SDS-PAGE to identify the optimal time point for maximal protein expression and cell harvest (step 2c).Do not express proteins for more than 3 days, because this can result in increased protein degradation and cell death.
The affinity capture of MBP-eIF4G with amylose resin is poor (Figure 1C) (step 4).Increasing the volume of resin does not lead to an increased capture of MBP-eIF4G, indicating that a fraction of MBP in expressed proteins is inaccessible for binding to amylose.We have tested different buffer compositions to improve affinity capture and concluded that buffer with high salt improves affinity capture and subsequent purification steps.Critically, SEC in buffer with lower salt (< 300 mM KCl) results in significant loss of eIF4G due to non-specific binding of eIF4G to the matrix of the column.
Potential solution eIF4E irreversibly aggregates at high concentrations.This is already observed in cells where eIF4E is predominantly insoluble.Use of solubilizing tag, such as MBP, increases the yield of soluble eIF4E (Figure 2C).In addition, expression of eIF4E in bacteria at 18 C-20 C increases solubility.To prevent the aggregation of eIF4E during purification and at any concentration step, supplement the buffer with 100 mM m 7 GTP sodium salt.Do not concentrate eIF4E above the concentration of supplemented m 7 GTP sodium salt.If the concentration of eIF4E after affinity capture on m 7 GTP resin is too high, some eIF4E may aggregate.However, these aggregates can be separated by SEC (Figure 2D) (step 12).

Problem 3
Yield of synthesized mRNA is low.

Potential solution
Use purified PCR products for in vitro transcription reactions (step 14c).Make sure that there are no precipitates in the 103 Reaction Buffer or 23 NTP/CAP solutions (step 15a).Typically, a pellet should appear after lithium chloride precipitation (step 17c).If not, add more lithium chloride and place at À20 C for at least another 1 h before repeating step 17c.

Problem 4
Difficulty in reconstituting condensates.

Potential solution
The concentrations detailed in step 26 should result in the assembly of condensates (Figure 5D), irrespective of the incubation temperature.Given that condensates can adsorb to plastic surfaces, use low binding tubes when possible.In addition, when transferring condensates to an imaging plate, pipet gently because condensates (especially non-heated condensates) are susceptible to shear stress and dissolution by pipetting.In general, remove unnecessary transfer steps when dealing with condensates.

Problem 5
Yeast extracts are poorly active and RLU measurements are low.

Potential solution
Make sure that none of the buffers and reagents contain EDTA.Extracts prepared from cells that are not in exponential phase can be less active.Harvest cells that are exponentially growing in YPD with an optical density A 600 0.8-1.2(step 19b).Low RLU values may also indicate reduced activity of CPK in the IVT reaction mix.Use IVT reaction mix that is less than 3 months old for optimal results.In addition, synthesized mRNA can aggregate when stored for longer than 6 months at À80 C. If

Figure 1 .
Figure 1.Purification of eIF4G and Pab1p (A) Schematic of MBP-eIF4G and MBP-Pab1p fusion protein constructs.The MBP is cleaved with HRV 3C protease.(B) Steps for the purification of eIF4G and Pab1p.(C) Coomassie stained 10% acrylamide gel of the purification steps of eIF4G.(D) Coomassie stained 10% acrylamide gel of the purification steps of Pab1p.(E) SEC chromatogram of eIF4G purification.Absorbance at 280 and 254 nm (A 280 and A 254 respectively) are shown.The elution peak with purified eIF4G is indicated.(F) SEC chromatogram of Pab1p purification.A 280 and A 254 are shown.The elution peak with purified Pab1p is indicated.

2 .
Express MBP-eIF4G and MBP-Pab1p in Sf9 cells.a. Prepare 1 L of Sf9 cells in ESF AF Insect Cell Culture Medium at a cell density of 2 million cells/mL.b.Infect cells with 10 mL of P2 baculovirus containing recombinant DNA for the expression of MBP-eIF4G or MBP-Pab1p (step 1n).c.Incubate infected Sf9 cells at 27 C with shaking at 100 rpm for 65 h.Note: For optimal expression, we recommend monitoring the expression of MBP-eIF4G and MBP-Pab1p at different time points following infection (see troubleshooting problem 1).

Figure 2 .
Figure 2. Purification of eIF4E (A) Schematic of MBP-eIF4E fusion protein construct.The MBP is cleaved with HRV 3C protease.(B) Steps for the purification of eIF4E.(C) Coomassie stained 15% acrylamide gel of the purification steps of eIF4E.(D) SEC chromatogram of eIF4E purification.A 280 and A 254 are shown.The elution peak with purified eIF4E is indicated.
c. Place resin into an empty 20 mL Econo-Pac Chromatography Column.d.Wash resin with 10 column volumes (CV) of Buffer E. e. Wash resin with 10 column volumes (CV) of Buffer E supplemented with 100 mM GTP to remove non-specific binders from the resin.11.Cleave MBP.a. Incubate the resin with Buffer E supplemented with 0.01 mg/mL of HRV 3C protease for 1 h at $23 C to cleave MBP.b.Wash resin with 10 CV of buffer E to remove cleaved MBP.c. Elute eIF4E from the resin with 4 CV of Buffer E supplemented with 100 mM m 7 GTP sodium salt.d.Measure protein concentration in elution.

Note:
Please refer to the manufacturer's protocol (Poly(A) Tailing Kit).Alternative kits can be used.a. Combine reagents in the following order at $23 C: b.Mix reaction by carefully pipetting up and down.

17.
Precipitate poly(A) tailed mRNA.a. Add 100 mL of 7.5 M lithium chloride.b.Place at À20 C for at least 1 h to $16 h to precipitate the mRNA.c.Centrifuge at 21,000 3 g for 20 min at 4 C. d.Wash pellet with 1 mL of cold 70% ethanol.e. Centrifuge at 21,000 3 g for 10 min at 4 C. f.Remove as much liquid as possible and dry the pellet at $23 C for 15-20 min until the pellet becomes translucent.g.Resuspend pellet in 100 mL of nuclease-free water.h.Centrifuge at 21,000 3 g for 5 min at $23 C to remove any mRNA aggregates.i. Transfer the supernatant to a new tube.j.Measure mRNA concentration.
18. Visualize synthesized mRNA using an RNA gel.a. Combine 2 mL of synthesized mRNA with 2 mL of 23 RNA loading dye.b.Heat at 95 C for 2 min.c.Place directly on ice.d.Load 4 mL of mRNA in 2% agarose gel in 13 TBE.e. Load 2 mL of RiboRuler High Range RNA Ladder in neighboring well.f.Run gel at 200 V for 10 min.g.Visualize mRNA with UV light (Figure3C).

Figure 4 .
Figure 4. Steps for the preparation of cell-free yeast extracts a. Harvest cells by centrifugation at 3,500 3 g for 5 min at 4 C. b.Resuspend cell pellet with 30 mL of cold Buffer Y/M.c. Harvest cells by centrifugation at 2,000 3 g for 4 min at 4 C. d.Wash cell pellet with 30 mL of cold Buffer Y/M.e. Repeat steps 20c-d three more times.
Place ATP, GTP, creatine phosphate, CPK, amino acids, tRNA and RNaseOUT on ice.Prepare the IVT reaction mix on ice and store 100-200 mL aliquots in 1.5 mL tubes at À80 C. * Prepare CPK stocks as detailed below.
Creatine phosphokinase-10 U/mL 110 mg (35,000 U)Store at À80 C for up to 1 year.
b. Add transfection mix to a 24-well plate together with 200 mL of 5 3 10 6 Sf9 cells/mL stock (this will result in 1 3 10 6 cells/mL).Determine cell density with Countess Cell Counting Chamber Slides and a Countess II Automated Cell Counter.c. Cover plate with a Breather-Easy membrane.d.Seal membrane and plate with parafilm.e. Place sealed plate at 27 C with shaking at 200 rpm for $16 h.
We recommend the Nano-Glo Luciferase Assay System to measure luminescence.a.Thaw IVT reactions on ice.b.Thaw the Nano-Glo Luciferase Assay buffer at $23 C. c. Prepare a mixture of Nano-Glo Luciferase Assay Buffer and Substrate to a final buffer:substrate volume ratio of 50:1.d.Add an equal volume of Nano-Glo Luciferase Assay Buffer-Substrate mixture to the IVT reactions.e.Transfer 5 mL of IVT reaction with Nano-Glo Luciferase Assay Buffer-Substrate mixture to a 384-well Low Volume White Round Bottom Polystyrene NBS Microplate.Note: Use white plates for luminescence measurements to maximize light output signal.To prevent crosstalk between adjacent wells, place samples in alternating wells.f.Measure luminescence using a Spark multimode microplate reader.If RLU measurements are low, refer to troubleshooting problem 5. i. Measure luminescence signal (counts) between 440-475 nm (maxima of light output) with a 1 s integration time.ii.If luminescence measurements exceed the detection limit of the detector, utilize attenuation filters or dilute the sample as needed.g.Clean the microplate with water and 20% ethanol and dry at $23 C. The plate can be re-used for future measurements. Note: